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Human Cytokine Array Q5

Details for Product No. ABIN625729, Supplier: Log in to see
Reactivity
Human
Methode Type
Sandwich ELISA
Application
Antibody Array (AA), Multiplex ELISA (mpELISA)
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Purpose Quantibody® Human Cytokine Array 5, multiplex ELISA array Kit for quantitative measurement of 40 Cytokines in 8-10 samples. Suitable for all sample Types
Brand Quantibody®
Sample Type Serum, Plasma, Cell Culture Supernatant, Cell Lysate, Tissue Lysate, Biological Fluids
Detection Method Fluorometric
Specificity Quantibody® Human Cytokine Array 5 detects: Adiponectin (ACRP30),Adipsin (Complement Factor D),Alpha-fetoprotein,ANGPTL4,Beta-2 Microglobulin,BCAM,CA125,CA15-3,CEA,CRP (C-Reactive Protein),ErbB2,Ferritin,FSH,GRO alpha (CXCL1),HCG beta,IGF-1 R,IL-1 R2,IL-3,IL-18 R beta (AcPL),IL-21,Leptin,MMP-1,MMP-2,MMP-3,MMP-8,MMP-9,MMP-10,MMP-13,NCAM-1 (CD56),Nidogen-1,NSE,Oncostatin M,Procalcitonin,Prolactin,PSA-free,Siglec-9,TACE,Thyroglobulin,TIMP-4,TSH
Components - Quantibody® Array Slide
- Blocking Buffer
- Wash Buffer 1
- Wash Buffer 2
- Lyophilized Standard Mix
- Biotinylated Detection Antibody Cocktail
- Streptavidin-Conjugated Fluor
- 30 ml Centrifuge Tube
- Adhesive Plastic Strips
- Manual
Material not included Distilled or deionized water
Small plastic boxes or containers
Pipettors, pipette tips and other common lab consumables
Orbital shaker or oscillating rocker
Aluminum foil
Gene microarray scanner or similar laser fluorescence scanner
Background Cytokines play an important role in innate immunity, apoptosis, angiogenesis, cell growth and differentiation. They are involved in interactions between different cell types, cellular responses to environmental conditions, and maintenance of homeostasis. In addition, cytokines are also involved in most disease processes, including cancer and cardiac diseases. The traditional method for cytokine detection and quantification is through the use of an enzyme-linked immunosorbent array (ELISA). In this method, target protein is first immobilized to a solid support. The immobilized protein is then complexed with an antibody that is linked to an enzyme. Detection of the enzyme-complex can then be visualized through the use of a substrate that produces a detectable signal. While the traditional method works well for a single protein, the overall procedure is time consuming and requires a lot of sample. With little sample to work with, conservation of precious small quantities becomes a risky task. Take the advantage of advancement in microarray technology over the last decade, more and more choices are available to the scientist today. A long-standing leader in the field, Raybiotech, has pioneered the development of cytokine antibody arrays, which has now been widely applied in the research community with hundreds of peer reviewed publications such as in Cell and Nature. Quantibody® array, our quantitative array platform, uses the multiplexed sandwich ELISA-based technology and enables researchers to accurately determine the concentration of multiple cytokines simultaneously. It combines the advantages of the high detection sensitivity / specificity of ELISA and the high throughput of the arrays. Like a traditional sandwich- based ELISA, it uses a pair of cytokine specific antibodies for detection. A capture antibody is first bound to the glass surface. After incubation with the sample, the target cytokine is trapped on the solid surface. A second biotin- labeled detection antibody is then added, which can recognize a different isotope of the target cytokine. The cytokine-antibody-biotin complex can then be visualized through the addition of the streptavidin-labeled Cy3 equivalent dye using a laser scanner. Unlike the traditional ELISA, Quantibody products use array format. By arraying multiple cytokine specific capture antibodies onto a glass support, multiplex detection of cytokines in one experiment is made possible. In detail, one standard glass slide is spotted with 16 wells of identical cytokine antibody arrays. Each antibody, together with the positive controls is arrayed in quadruplicate. The slide comes with a 16-well removable gasket which allows for the process of 16 samples in one slide. Four slide chips can be nested into a tray, which matches a standard microplate and allows for automated robotic high throughput process of 64 arrays simultaneously. For cytokine quantification, the array specific cytokine standards, whose concentration has been predetermined, are provided to generate a standard curve for each cytokine. In a real experiment, standard cytokines and samples will be assayed in each array simultaneously through a sandwich ELISA procedure. By comparing signals from unknown samples to the standard curve, the cytokine concentration in the samples will be determined. Quantibody® array kits have been confirmed to have similar detection sensitivity as traditional ELISA. Our current high density Quantibody kits allow scientists to quantitatively determine the concentration of 240 human or 120 mouse cytokines in a single experiment. This is not only one of the most efficient products on the market for cytokine quantification, but makes it more affordable for quantification of large number of proteins. Simultaneous detection of multiple cytokines undoubtedly provides a powerful tool for drug and biomarker discovery.
Application Notes Completely cover array area with sample or buffer during incubation. Avoid foaming during incubation steps. Perform all incubation and wash steps under gentle rocking or rotation. Cover the incubation chamber with adhesive film during incubation, particularly when incubation is more than 2 hours or <70 µl of sample or reagent is used. Several incubation steps such as step 6 (blocking), step 7 (sample incubation), step 10 (detection antibody incubation), or step 13 (Cy3 equivalent dyestreptavidin incubation) may be done overnight at 4°C. Please make sure to cover the incubation chamber tightly to prevent evaporation.
Comment

The Quantibody arrays are quantitative multiplex ELISA arrays featuring fluorescent detection. The antibodies are spotted on glass slide solid supports and require a laser scanner for data collection. Cytokine standards are provided with the array for calculation of target protein concentrations. All Quantibody arrays feature the sandwich immunoassay principle, utilizing an immobilized capture antibody along with a corresponding biotinylated detection antibody.

Sample Volume 50 μL
Assay Time 6 h
Plate Glass Slide
Protocol - Dry the glass slide
- Prepare Standards
- Block array surface
- Incubate with Samples and Standards
- Incubate with Biotinylated Detection Antibody Cocktail
- Incubate with Streptavidin-Conjugated Fluor
- Disassemble the glass slide
- Scan with a gene microarray laser scanner
- Perform densitometry and analysis
Sample Preparation

Use serum-free conditioned media if possible. If serum-containing conditioned media is required, it is highly recommended that complete medium be used as a control since many types of sera contains cytokines. We recommend the following parameters for your samples: 50 to 100 l of original or diluted serum, plasma, cell culture media, or other body fluid, or 50-500 g/ml of protein for cell and tissue lysates. If you experience high background or the readings exceed the detection range, further dilution of your sample is recommended.

Assay Procedure
  1. Take out the glass slide from the box, and let it equilibrate to room temperature inside the sealed plastic bag for 20-30 minutes. Remove slide from the plastic bag, peel off the cover film, and let it air dry for another 1-2 hours.
    2. Reconstitute the Cytokine Standard Mix (lyophilized) by adding 500 µl Sample Diluent to the tube. For best recovery, always quick-spin vial prior to opening. Dissolve the powder thoroughly by a gentle mix. Labeled the tube as Std1.
    3. Label 6 clean microcentrifuge tubes as Std2 to Std7. Add 200 µl Sample Diluent to each of the tubes.
    4. Pipette 100 µl Std1 into tube Std2 and mix gently. Perform 5 more serial dilutions by adding 100 µl Std2 to tube Std3 and so on.
    5. Add 100 µl Sample Diluent to another tube labeled as CNTRL. Do not add standard cytokines or samples to the CNTRL tube, which will be used as negative control. For best results, include a set of standards in each slide.
    6. Add 100 µl Sample Diluent into each well and incubate at room temperature for 30 minutes to block slides.
    7. Decant buffer from each well. Add 100 µl standard cytokines or samples to each well. Incubate arrays at room temperature for 1-2 hour.
    8. Wash:
    - Decant the samples from each well, and wash 5 times (5 min each) with 150 µl of 1X Wash Buffer I at room temperature with gentle rocking. Completely remove wash buffer in each wash step. Dilute 20x Wash Buffer I with H2O.
    - Decant the 1x Wash Buffer I from each well, wash 2 times (5 min each) with 150 µl of 1X Wash Buffer II at room temperature with gentle rocking. Completely remove wash buffer in each wash step. Dilute 20X Wash Buffer II with H2O.
    9. Reconstitute the detection antibody by adding 1.4 ml of Sample Diluent to the tube. Spin briefly.
    10. Add 80 µl of the detection antibody cocktail to each well. Incubate at room temperature for 1-2 hour.
    11. Decant the samples from each well, and wash 5 times (5 mins each) with 150 µl of 1X Wash Buffer I and then 2 times with 150 µl of 1x Wash Buffer II at room temperature with gentle rocking. Completely remove wash buffer in each wash step.
    12. After briefly spinning down, add 1.4 ml of Sample Diluent to Cy3 equivalent dye-conjugated streptavidin tube. Mix gently.
    13. Add 80 µl of Cy3 equivalent dye-conjugated streptavidin to each well. Cover the device with aluminum foil to avoid exposure to light or incubate in dark room. Incubate at room temperature for 1 hour.
    14. Decant the samples from each well, and wash 5 times (5 mins each) with 150 µl of 1X Wash Buffer I at room temperature with gentle rocking. Completely remove wash buffer in each wash step.
    15. Disassemble the device by pushing clips outward from the slide side. Carefully remove the slide from the gasket.
    16. Place the slide in the Slide Washer/Dryer (a 4-slide holder/centrifuge tube), add enough 1x Wash Buffer I (about 30 ml) to cover the whole slide, and then gently shake at room temperature for 15 minutes. Decant Wash Buffer I. Wash with 1x Wash Buffer II (about 30 ml) and gently shake at room temperature for 5 minutes.
    17. Remove water droplets completely by gently applying suction with a pipette to remove water droplets. Do not touch the array, only the sides.
    18. Imaging: The signals can be visualized through use of a laser scanner equipped with a Cy3 wavelength (green channel) such as Axon GenePix. Make sure that the signal from the well containing the highest standard concentration (Std1) receives the highest possible reading, yet remains unsaturated.
Calculation of Results

Data extraction can be done using the GAL file that is specific for this array along with the microarray analysis software (GenePix, ScanArray Express, ArrayVision, MicroVigene, etc.).

Assay Precision Reproducibility: CV < 20%
Restrictions For Research Use only
Handling Advice Do not touch the surface of the slides, as the microarray slides are very sensitive. Hold the slides by the edges only. Handle all buffers and slides with powder free gloves. Handle glass slide/s in clean environment. The Quantibody slides do not have bar codes. To help distinguish one slide from another, transcribe the slide serial number from the slide bag to the back of the slide with an ultra-fine point permanent marker. Please Note:Red permanent marker can significantly interfere with fluorescent signal detection. We recommend marking your slides with a green, blue or black ultra-fine point permanent marker. Please write the number on the very bottom edge of the slide. Do not write on the arrayed well areas.
Storage -20 °C
Storage Comment Upon receipt, all components should be stored at -20°C. The kit will retain activity for up to 6 months. Once thawed, the glass slide, standard mix, antibody cocktail and dye-conjugated Streptavidin should be kept at -20°C. All other components may be stored at 4°C. The entire kit should be used within 6 months of purchase.
Expiry Date 6 months
Product cited in: Bartolini, Inzitari, Castagnoli et al.: "Correlation of isotopic cisternographic patterns in multiple sclerosis with CSF IgG values." in: Annals of neurology, Vol. 12, Issue 5, pp. 486-9, 1983 (PubMed).